The epicardium, an epithelial sheet of specialized mesothelium that covers the heart, is gaining recognition as an important source of signals and cells that modulate heart development and postnatal heart function. In the developing heart, epicardial cells adopt a mesenchymal phenotype and migrate into the myocardium, differentiating into interstitial and coronary vascular cells. Signals secreted from developing epicardium also modulate the growth and differentiation of underlying myocardium. The transcription factor Wt1 is expressed in the epicardium, but not myocardium or endocardium. The epicardium of Wt1 knockout hearts is largely intact, but embryos develop heart failure due to myocardial hypoplasia. Using Wt1-driven Cre expression to follow the fate of epicardial cells, we found that epicardium differentiates into cardiomyocytes during normal heart development. Consistent with this differentiation potential, we found that the epicardium originates from precursors that express Isl1 and Nkx2-5, markers of cardiac progenitor cells. The overall goal of this proposal is to further define the contribution of epicardium to the developing and postnatal heart. In Aim 1, we test the hypothesis that Wt1+ epicardial cells are multipotent and able to differentiate into cardiomyocyte, smooth muscle, and endothelial fates. In Aim 2, we test the hypothesis that postnatal epicardium contributes to the myocardial wall in the normal heart and after myocardial infarction. In Aim 3, we expand upon our preliminary observations that the gene encoding the key retinoic acid synthesizing enzyme Raldh2 is genetically downstream of Wt1. We test the hypothesis that Raldh2 is directly regulated by Wt1. These experiments will characterize a novel cardiac progenitor population and inform us of its contributions of the developing and postnatal heart. These data will add to the growing interest in the epicardium as a source of cells and trophic factors for regenerative medicine. PUBLIC HEALTH RELEVANCE: The epicardium is a sheet of cells that covers heart muscle tissue. Recently, there has been increased interest in epicardium because of its role in forming the blood vessels of the fetal heart, and the potential that this activity can be reactivated in the mature heart. We have found that epicardium can also form heart muscle cells. The overall goal of this proposal is to define the cells and signals that the epicardium contributes to the developing and postnatal heart. These experiments will prepare the foundation for future studies on potential therapeutic uses of epicardium as a source of cells and signals to treat heart failure.